Anne Mullen

Anti-inflammatory effects of EPA and DHA are dependent upon time and dose-response elements associated with LPS stimulation in THP-1-derived macrophages

The long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) of fish oil, eicosapentanoic (EPA) and docosahexanoic (DHA) acids are considered cardioprotective. Inflammation elicited by macrophages is increasingly recognised in the aetiology of metabolic syndrome. This study investigated the differential anti-inflammatory potential of EPA and DHA through cytokine production and nuclear factor (NF)-kappaB signalling in a human macrophage model. We investigated the dependency of LC n-3 PUFA immune-modulation on concentration and duration of lipopolysaccharide (LPS) activation. Interleukin (IL)-1beta, IL-6 and tumor necrosis factor-alpha secretion from EPA, DHA and control cells were differentially limited by LPS concentration. In all cases, there was no benefit in activating cells with >0.1 microg/ml LPS. LC n-3 PUFA decreased proinflammatory cytokines production, an effect modulated by LPS concentration. Expression of the transcription factor NF-kappaB p65 was significantly reduced in the nucleus and retained in the cytoplasm of EPA- and/or DHA-treated macrophages during 5-h activation with 0.1 microg/ml LPS. Nuclear binding of p65 was significantly reduced in EPA- and DHA-treated cells at 2-h LPS activation. Over the time course, expression of nuclear IkappaBalpha was significantly reduced, cytoplasmic NF-kappaB p50 significantly increased and cytoplasmic cleaving enzyme IkappaB inhibitor complex significantly reduced in LC n-3 PUFA-treated cells. EPA and DHA down-regulated the production of proinflammatory cytokines associated with the aetiology of metabolic syndrome, NF-kappaB transcriptional activity and upstream cytoplasmic signalling events. Immune responses are dynamic, and the present study suggests a nutrient sensitive window of LPS activation at which EPA and DHA are strongly anti-inflammatory.

Effect of dietary supplementation with conjugated linoleic acid on markers of calcium and bone metabolism in healthy adult men

Introduction:Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism in experimental animals and cells in culture, but there are limited human data available.Objective:To investigate the effect of CLA supplementation on biomarkers of calcium and bone metabolism in healthy adult males.Design:The study consisted of a double-blind, placebo-controlled trial in which 60 healthy adult males (aged 39–64 y) were randomly assigned to receive daily either 3.0 g CLA isomer blend (50:50% cis-9,trans-11:trans-10,cis-12 isomers) or a palm/bean oil blend (placebo) for 8 weeks. Urine and blood samples were collected at weeks 0 and 8 and were analysed for biomarkers of calcium and bone metabolism.Results:Supplementation with CLA or placebo for 8 weeks had no significant effects on markers of bone formation (serum osteocalcin and bone-specific alkaline phosphatase) or bone resorption (serum C-telopeptide-related fraction of type 1 collagen degradation products, urinary N-telopeptide-related fraction of type 1 collagen degradation products, urinary pyridinoline and deoxypyridinoline), or on serum or urinary calcium levels. Baseline levels of these biochemical parameters were similar in both groups of subjects. While the placebo had no effect, CLA supplementation resulted in a three-fold increase (P<0.00001) in cis-9,trans-11 CLA isomer in total plasma lipids.Conclusion:Under the conditions tested in this double-blind, placebo-controlled trial in adult men, a CLA supplement of mixed isomers did not affect markers of calcium or bone metabolism. Further investigation of the effects of CLA on calcium and bone metabolism in other gender- and age-groups is warranted.Sponsorship:Irish Government.

Obesity and the gastrointestinal microbiota: a review of associations and mechanisms

The two-way obesity model that considers only the interplay between humans and their environment has been revised to include the gastrointestinal microbiota. Notable perturbations in the bacterial communities in obese individuals have been uncovered. Research is helping to distinguish between the obesogenic mechanisms attributable to diet and those that may be associated with the microbiota. Examples include studies in which transplant of the microbiota from murine models of weight loss (gastric bypass) into germ-free mice resulted in significant weight loss. Several mechanisms have been identified that suggest the microbiota may play a role in obesity development and propagation. There is some evidence from animal and human studies that the microbiota in the obese harvests energy more effectively and may manipulate host gene function leading to increased adiposity, aggravation of inflammatory mechanisms, metabolic endotoxemia, and metabolic dysfunction. Research findings highlight the potential of the microbiota to influence body weight and they allude to its potential therapeutic use in tackling the costly global epidemic of obesity.

Eicosapentaenoic acid and 3,10 dithia stearic acid inhibit the desaturation of trans-vaccenic acid into cis-9, trans-11-conjugated linoleic acid through different pathways in Caco-2 and T84 cells

Stearoyl-CoA desaturase (SCD) is a key enzyme that determines the composition and metabolic fate of ingested fatty acids, in particular the conversion of trans-vaccenic acid (TVA) to conjugated linoleic acid (CLA). The present study addressed the hypothesis that intestinal TVA absorption and biotransformation into CLA can be modulated by EPA and 3,10-dithia stearic acid (DSA) via altered SCD mRNA levels and desaturation indices (cis-9, trans-11-CLA:TVA and oleic acid:stearic acid ratios) in Caco-2 and T84 cells, two well-established in vitro models of the human intestinal epithelium. The study determined the effect of acute (3 h with 0.3 mm-EPA or 0.3 mm-DSA) and acute-on-chronic (1 week with 0.03 mm-EPA or -DSA, followed by respectively, 0.3 mm-EPA or -DSA for 3 h) treatments. In both cell lines, acute EPA treatment did not alter SCD desaturation indices, whereas the acute-on-chronic treatment affected these surrogate markers of SCD activity. This was associated with reduced sterol regulatory-element binding protein-1c and SCD mRNA levels. In contrast, acute and acute-on-chronic DSA treatments significantly reduced SCD desaturation indices without affecting SCD mRNA levels in Caco-2 cells. The present study on intestinal cells shows that the conversion rate of TVA to c9, t11-CLA is affected by other fatty acids present in the diet such as EPA, confirming previous observations in hepatic and mammary cell models.

A Micronutrient-Fortified Food Enhances Iron and Selenium Status of Zambian Infants but Has Limited Efficacy on Zinc

Micronutrient-fortified, cereal-based infant foods are recommended for reducing multiple micronutrient deficiencies in low-income countries, but their nutritional quality is not always optimal. In a double-blind randomized trial, we compared the efficacy of a locally produced porridge based on maize, beans, bambaranuts, and groundnuts fortified with 19 (rich) or 9 (basal) micronutrients. Infants aged 6 mo from Lusaka, Zambia were randomized to receive the richly fortified (n = 373) or basal (n = 370) porridge daily for 12 mo along with routine vitamin A supplements. Baseline and final micronutrient status and inflammation (based on α-1-glycoprotein) were assessed using nonfasting blood samples. Baseline prevalence of anemia (39%) and zinc deficiency (51%) were a public health concern. There were overall treatment effects on hemoglobin (Hb) (P = 0.001), serum transferrin receptor (P < 0.001), serum ferritin (P < 0.001), and serum selenium (P = 0.009); biomarker responses for iron and zinc were modified by baseline concentrations, and for Hb and iron by socioeconomic status. At 18 mo, the adjusted odds of anemia, iron deficiency anemia (Hb <105 g/L and transferrin receptor > 11.0 mg/L), and iron deficiency were 0.37 (95% CI = 0.25, 0.55), 0.18 (0.09, 0.35), and 0.30 (0.18, 0.50) times those in the basal group, respectively. The rich level of fortification had no overall treatment effect on serum zinc (1.09; 0.66, 1.80) but improved serum zinc in children with lower Hb concentrations at baseline (P = 0.024). A locally produced cereal- and legume-based infant food richly fortified with micronutrients reduced anemia and improved iron and selenium status but may require reformulation to improve the biochemical zinc status of urban Zambian infants.

The effects of micronutrient-fortified complementary/replacement food on intestinal permeability and systemic markers of inflammation among maternally HIV-exposed and unexposed Zambian infants

The present randomised trial investigated the effects of feeding Zambian infants from 6 to 18 months old either a richly or basal micronutrient-fortified complementary/replacement food on gut integrity and systemic inflammation. Blood samples were obtained from all infants (n 743) at 6 and 18 months for the assessment of serum C-reactive protein (CRP) and α1-acid glycoprotein (AGP). A subsample of 502 infants, selected from the main cohort to include a larger proportion of infants with HIV-positive mothers, was assigned to lactulose/mannitol gut permeability tests. Lactulose:mannitol (L:M) ratio analyses were adjusted for baseline urinary L:M ratio, socio-economic status, mothers education, season of birth and baseline stunting, and stratified by maternal antenatal HIV status, childs sex, concurrent breast-feeding status and anaemia at baseline. There was no significant difference in geometric mean L:M ratio between the richly fortified and basal-fortified porridge arms at 12 months (0·47 (95 % CI 0·41, 0·55) v. 0·41 (95 % CI 0·34, 0·49); P = 0·16 adjusted). At 18 months, the richly fortified porridge group had a significantly higher geometric mean L:M ratio than the basal-fortified group (0·23 (95 % CI 0·19, 0·28) v. 0·15 (95 % CI 0·12, 0·19); P = 0·02 adjusted). This effect was evident for all stratifications, significantly among boys (P = 0·04), among the infants of HIV-negative mothers (P = 0·01), among the infants of HIV-negative mothers not concurrently breast-fed (P = 0·01) and among those who were not anaemic at baseline (P = 0·03). CRP, but not AGP, was positively associated with L:M ratio, but there were no significant effects of the diet on either CRP or AGP. In conclusion, a richly fortified complementary/replacement food did not benefit and may have worsened intestinal permeability.

Microarray analysis reveals altered lipid and glucose metabolism genes in differentiated, ritonavir-treated 3T3-L1 adipocytes

OBJECTIVE HIV lipodystrophy is characterised by abnormal adipose tissue distribution and metabolism, as a result of altered adipocyte function and gene expression. The protease inhibitor ritonavir is associated with the development of lipodystrophy. Quantifying changes in adipogenic gene expression in the presence of ritonavir may help to identify therapeutic targets for HIV lipodystrophy. METHODS Affymetrix Mouse Genome 430 2.0 oligonucleotide microarray was used to investigate gene expression in 3T3-L1 adipocytes treated with 20 µmol/l ritonavir or vehicle control (ethanol). Pparg, Adipoq, Retn and Il6 expression were validated by real time RT-PCR. Transcriptional signalling through PPAR-γ was investigated using a DNA-binding ELISA. Changes in adipocyte function were investigated through secreted adiponectin quantification using ELISA and Oil Red O staining for triglyceride storage. RESULTS Expression of 389 genes was altered by more than 5-fold in the presence of ritonavir (all P < 0.001). Gene ontology analysis revealed down-regulation of genes responsible for adipocyte triglyceride accumulation including complement factor D (Cfd; 238.42-fold), Cidec (73.75-fold) and Pparg (5.63-fold). Glucose transport genes were also down-regulated including Adipoq (24.42-fold) and Glut4 (13.36-fold), while Il6 was up-regulated (10.39-fold). PPAR-γ regulatory genes Cebpa (11.33-fold) and liver-X-receptor α (Nr1h3) were down-regulated. Changes in Pparg, Adipoq and Il6 were confirmed by RT-PCR. PPAR-γ binding to its nuclear consensus site, adiponectin secretion and triglyceride accumulation were all reduced by ritonavir. CONCLUSION Ritonavir had a significant effect on expression of genes involved in adipocyte differentiation, lipid accumulation and glucose metabolism. Down-regulation of Pparg may be mediated by changes in Cebpa, Lcn2 and Nr1h3.

Conjugated Linoleic Acid Isomers Exert Differential Effects on an Adipocyte Model of HIV-associated Lipodystrophy

BACKGROUND HIV-associated lipodystrophy is associated with decreased expression of PPAR-γ in adipose tissue. Conjugated linoleic acid (CLA) isomers (cis9, trans11 and trans10, cis12) are putative PPAR-γ agonists, but have not previously been investigated in the context of HIVassociated lipodystrophy. METHOD 3T3-L1 pre-adipocytes were differentiated in the presence of ritonavir (20 μM as per previous experimental models) and 100 μM cis9,trans11, trans10,cis12 or vehicle control, DMSO. Microarray analysis, RT-PCR, DNA binding ELISA and Oil Red O staining were used to investigate adipocyte gene expression and binding, protein secretion and triglyceride storage. RESULTS trans10, cis12 + ritonavir altered the expression of 2160 gene transcripts greater than 1.5-fold compared with control, while 257 gene transcripts were altered by cis9,trans11 + ritonavir (P<0.001). trans10,cis12 + ritonavir down-regulated Pparg (-1.55) and Adipoq (-2.95), as well as differentiation (Fcor (-4.78-fold), Arl4a (-4.84), Itga6 (-2.45), Id4 (-2.01)) and triglyceride storage genes (Mrap (- 8.25), Scd1 (-4.34), Lipin1 (-2.52)). Changes in Adipoq were confirmed by RT-PCR (P=0.038) and adiponectin secretion by ELISA (P= 0.003). cis9,trans11 + ritonavir increased PPAR-γ nuclear binding to its gene response element (P=0.038). Both isomers increased triglyceride storage in the presence of ritonavir (P<0.001). CONCLUSION In the presence of ritonavir, trans10, cis12 appears to be detrimental, while cis9, trans11 was beneficial and may mediate its effects via PPAR-γ. Further research is required to determine the potential role of CLA isomers as therapeutic agents in the management of HIV-associated lipodystrophy.